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Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB.

Min HK, Kim SM, Baek SY, Woo JW, Park JS, Cho ML, Lee J, Kwok SK, Kim SW, Park SH - PLoS ONE (2015)

Bottom Line: AEBS decreased Th17 cell numbers in spleen of CIA mice.The anti-arthritic effects of AEBS were associated with decreases in Th17 cell numbers, and the levels of proinflammatory cytokines synthesized by such cells, mediated via suppression of NF-κB signaling.Additionally, AEBS suppressed osteoclastogenesis and reduced oxidative stress levels.

View Article: PubMed Central - PubMed

Affiliation: Rheumatism Research Center, Catholic Research Institute of Medical Science, The Catholic University of Korea, Seoul, South Korea.

ABSTRACT

Introduction: Oxidative stress plays a role in the pathogenesis of rheumatoid arthritis (RA). Anthocyanin is a plant antioxidant. We investigated the therapeutic effects of anthocyanin extracted from black soybean seed coats (AEBS) in a murine model of collagen-induced arthritis (CIA) and human peripheral blood mononuclear cells (PBMCs) and explored possible mechanisms by which AEBS might exert anti-arthritic effects.

Material and methods: CIA was induced in DBA/1J mice. Cytokine levels were measured via enzyme-linked immunosorbent assays. Joints were assessed in terms of arthritis incidence, clinical arthritis scores, and histological features. The extent of oxidative stress in affected joints was determined by measuring the levels of nitrotyrosine and inducible nitric oxide synthase. NF-κB activity was assayed by measuring the ratio of phosphorylated IκB to total IκB via Western blotting. Th17 cells were stained with antibodies against CD4, IL-17, and STAT3. Osteoclast formation was assessed via TRAP staining and measurement of osteoclast-specific mRNA levels.

Results: In the CIA model, AEBS decreased the incidence of arthritis, histological inflammation, cartilage scores, and oxidative stress. AEBS reduced the levels of proinflammatory cytokines in affected joints of CIA mice and suppressed NF-κB signaling. AEBS decreased Th17 cell numbers in spleen of CIA mice. Additionally, AEBS repressed differentiation of Th17 cells and expression of Th17-associated genes in vitro, in both splenocytes of naïve DBA/1J mice and human PBMCs. In vitro, the numbers of both human and mouse tartrate-resistant acid phosphatase+ (TRAP) multinucleated cells fell, in a dose-dependent manner, upon addition of AEBS.

Conclusions: The anti-arthritic effects of AEBS were associated with decreases in Th17 cell numbers, and the levels of proinflammatory cytokines synthesized by such cells, mediated via suppression of NF-κB signaling. Additionally, AEBS suppressed osteoclastogenesis and reduced oxidative stress levels.

No MeSH data available.


Related in: MedlinePlus

AEBS reduces Th17 cell numbers in CIA mice.Tissues were obtained and stained when the mice were sacrificed (8weeks from immunization). (A) AEBS reduced the expression levels of IL-17, IL-6, TNF-α, and IL-1β in synovial tissues. Tissue sections from joints of CIA mice given AEBS (n = 5) or vehicle (n = 5) were stained with anti-IL-17, anti-IL-6, anti-TNF-α, and anti-IL-1β antibodies; or anti-isotype control antibodies. Stained cells are brown in color. Original magnification ×400. The cells showing positive IL-17, IL-6, TNF- α, and IL-1β were enumerated visually at higher magnification (projected on a screen) by four individuals, and the mean values are presented (cells/field). (B) Pooled splenocytes from CIA mice given AEBS were cultured with PMA (25 ng/ml) and ionomycin (250 ng/ml) for 30 min and p-IκB levels determined via Western blotting. Data are expressed as means ± SDs. *P < 0.05 compared to the vehicle-treated group. Each experiment was performed 3 times.
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pone.0138201.g002: AEBS reduces Th17 cell numbers in CIA mice.Tissues were obtained and stained when the mice were sacrificed (8weeks from immunization). (A) AEBS reduced the expression levels of IL-17, IL-6, TNF-α, and IL-1β in synovial tissues. Tissue sections from joints of CIA mice given AEBS (n = 5) or vehicle (n = 5) were stained with anti-IL-17, anti-IL-6, anti-TNF-α, and anti-IL-1β antibodies; or anti-isotype control antibodies. Stained cells are brown in color. Original magnification ×400. The cells showing positive IL-17, IL-6, TNF- α, and IL-1β were enumerated visually at higher magnification (projected on a screen) by four individuals, and the mean values are presented (cells/field). (B) Pooled splenocytes from CIA mice given AEBS were cultured with PMA (25 ng/ml) and ionomycin (250 ng/ml) for 30 min and p-IκB levels determined via Western blotting. Data are expressed as means ± SDs. *P < 0.05 compared to the vehicle-treated group. Each experiment was performed 3 times.

Mentions: Expression of proinflammatory cytokines was assessed by staining affected joints with specific antibodies detecting IL-1β, IL-6, IL-17, and TNF-α, and enumerating the cell populations synthesizing such cytokines. To explore activation of NF-κB signaling, we measured the levels of IκB and p-IκB. AEBS-treated CIA mice had lower levels of IL-1β, IL-6, IL-17, and TNF-α in affected joints than did vehicle-treated mice, and the cell populations synthesizing such cytokines were also reduced in test animals (Fig 2A). The p-IκB:IκB ratio was non-significantly lower in the AEBS-treated group than in controls (Fig 2B). These results suggest that AEBS reduced inflammation of affected joints, the numbers of cells secreting proinflammatory cytokines, and trend to suppress NF-κB signaling.


Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB.

Min HK, Kim SM, Baek SY, Woo JW, Park JS, Cho ML, Lee J, Kwok SK, Kim SW, Park SH - PLoS ONE (2015)

AEBS reduces Th17 cell numbers in CIA mice.Tissues were obtained and stained when the mice were sacrificed (8weeks from immunization). (A) AEBS reduced the expression levels of IL-17, IL-6, TNF-α, and IL-1β in synovial tissues. Tissue sections from joints of CIA mice given AEBS (n = 5) or vehicle (n = 5) were stained with anti-IL-17, anti-IL-6, anti-TNF-α, and anti-IL-1β antibodies; or anti-isotype control antibodies. Stained cells are brown in color. Original magnification ×400. The cells showing positive IL-17, IL-6, TNF- α, and IL-1β were enumerated visually at higher magnification (projected on a screen) by four individuals, and the mean values are presented (cells/field). (B) Pooled splenocytes from CIA mice given AEBS were cultured with PMA (25 ng/ml) and ionomycin (250 ng/ml) for 30 min and p-IκB levels determined via Western blotting. Data are expressed as means ± SDs. *P < 0.05 compared to the vehicle-treated group. Each experiment was performed 3 times.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636296&req=5

pone.0138201.g002: AEBS reduces Th17 cell numbers in CIA mice.Tissues were obtained and stained when the mice were sacrificed (8weeks from immunization). (A) AEBS reduced the expression levels of IL-17, IL-6, TNF-α, and IL-1β in synovial tissues. Tissue sections from joints of CIA mice given AEBS (n = 5) or vehicle (n = 5) were stained with anti-IL-17, anti-IL-6, anti-TNF-α, and anti-IL-1β antibodies; or anti-isotype control antibodies. Stained cells are brown in color. Original magnification ×400. The cells showing positive IL-17, IL-6, TNF- α, and IL-1β were enumerated visually at higher magnification (projected on a screen) by four individuals, and the mean values are presented (cells/field). (B) Pooled splenocytes from CIA mice given AEBS were cultured with PMA (25 ng/ml) and ionomycin (250 ng/ml) for 30 min and p-IκB levels determined via Western blotting. Data are expressed as means ± SDs. *P < 0.05 compared to the vehicle-treated group. Each experiment was performed 3 times.
Mentions: Expression of proinflammatory cytokines was assessed by staining affected joints with specific antibodies detecting IL-1β, IL-6, IL-17, and TNF-α, and enumerating the cell populations synthesizing such cytokines. To explore activation of NF-κB signaling, we measured the levels of IκB and p-IκB. AEBS-treated CIA mice had lower levels of IL-1β, IL-6, IL-17, and TNF-α in affected joints than did vehicle-treated mice, and the cell populations synthesizing such cytokines were also reduced in test animals (Fig 2A). The p-IκB:IκB ratio was non-significantly lower in the AEBS-treated group than in controls (Fig 2B). These results suggest that AEBS reduced inflammation of affected joints, the numbers of cells secreting proinflammatory cytokines, and trend to suppress NF-κB signaling.

Bottom Line: AEBS decreased Th17 cell numbers in spleen of CIA mice.The anti-arthritic effects of AEBS were associated with decreases in Th17 cell numbers, and the levels of proinflammatory cytokines synthesized by such cells, mediated via suppression of NF-κB signaling.Additionally, AEBS suppressed osteoclastogenesis and reduced oxidative stress levels.

View Article: PubMed Central - PubMed

Affiliation: Rheumatism Research Center, Catholic Research Institute of Medical Science, The Catholic University of Korea, Seoul, South Korea.

ABSTRACT

Introduction: Oxidative stress plays a role in the pathogenesis of rheumatoid arthritis (RA). Anthocyanin is a plant antioxidant. We investigated the therapeutic effects of anthocyanin extracted from black soybean seed coats (AEBS) in a murine model of collagen-induced arthritis (CIA) and human peripheral blood mononuclear cells (PBMCs) and explored possible mechanisms by which AEBS might exert anti-arthritic effects.

Material and methods: CIA was induced in DBA/1J mice. Cytokine levels were measured via enzyme-linked immunosorbent assays. Joints were assessed in terms of arthritis incidence, clinical arthritis scores, and histological features. The extent of oxidative stress in affected joints was determined by measuring the levels of nitrotyrosine and inducible nitric oxide synthase. NF-κB activity was assayed by measuring the ratio of phosphorylated IκB to total IκB via Western blotting. Th17 cells were stained with antibodies against CD4, IL-17, and STAT3. Osteoclast formation was assessed via TRAP staining and measurement of osteoclast-specific mRNA levels.

Results: In the CIA model, AEBS decreased the incidence of arthritis, histological inflammation, cartilage scores, and oxidative stress. AEBS reduced the levels of proinflammatory cytokines in affected joints of CIA mice and suppressed NF-κB signaling. AEBS decreased Th17 cell numbers in spleen of CIA mice. Additionally, AEBS repressed differentiation of Th17 cells and expression of Th17-associated genes in vitro, in both splenocytes of naïve DBA/1J mice and human PBMCs. In vitro, the numbers of both human and mouse tartrate-resistant acid phosphatase+ (TRAP) multinucleated cells fell, in a dose-dependent manner, upon addition of AEBS.

Conclusions: The anti-arthritic effects of AEBS were associated with decreases in Th17 cell numbers, and the levels of proinflammatory cytokines synthesized by such cells, mediated via suppression of NF-κB signaling. Additionally, AEBS suppressed osteoclastogenesis and reduced oxidative stress levels.

No MeSH data available.


Related in: MedlinePlus